Characterization of Two Genes Encoding Putative Cysteine Synthase Required for Cysteine Biosynthesis in<i>Schizosaccharomyces pombe</i>

Fujita, Yuka; Takegawa, Kaoru

doi:10.1271/bbb.68.306

Cited by 28 publications

(23 citation statements)

References 21 publications

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“…1B, h). As expected, a cys1a mutant exhibited cysteine auxotrophy (Fujita & Takegawa, 2004). In contrast to the cysteine biosynthetic pathway, methionine biosynthesis in Sch.…”

Section: Introductionsupporting

confidence: 78%

“…CYS4 and CYS3 were co-expressed in the Sch. pombe cysteine auxotroph Dcys1a, which lacks the O-acetylserine pathway (Fujita & Takegawa, 2004). Co-expression of CYS4 and CYS3 in the cys1a mutant restored cysteine prototrophy, indicating functional expression in this strain (data not shown).…”

Section: Introduction Of the Cystathionine Pathway Into Met26 Disruptmentioning

confidence: 89%

“…The parent Sch. pombe strain ARC039 (Fujita & Takegawa, 2004) was provided by Asahi Glass Co. Ltd. Strain YF023 was generated through development of a new transformation system for Sch. pombe (Fujita et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

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Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

et al. 2006

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Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis, i.e. methylation of homocysteine. A search of the Schizosaccharomyces pombe genomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, and thus the gene was identified as a methionine synthase and designated met26. The met26 mutant was found to exhibit a remarkable growth defect in the absence of adenine even in medium supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeast Saccharomyces cerevisiae, which has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes from Sac. cerevisiae constituting the cystathionine pathway (CYS4 and CYS3) into Sch. pombe Δmet26 cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in Δmet26 cells was higher than in other methionine auxotrophs and that introduction of the Sac. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in the met26 mutant.

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“…1B, h). As expected, a cys1a mutant exhibited cysteine auxotrophy (Fujita & Takegawa, 2004). In contrast to the cysteine biosynthetic pathway, methionine biosynthesis in Sch.…”

Section: Introductionsupporting

confidence: 78%

Section: Introduction Of the Cystathionine Pathway Into Met26 Disruptmentioning

confidence: 89%

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Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

et al. 2006

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“…In S. cerevisiae, only some strains present detectable activities of the enzymes involved in the OAS pathway, cysteine being predominantly synthesized via the cystathionine intermediary (22). On the other hand, S. pombe synthesizes cysteine mainly through the OAS pathway, which makes its response to cadmium different from S. cerevisiae (5).…”

Section: Resultsmentioning

confidence: 99%

Transcriptome Analysis in Response to Heat Shock and Cadmium in the Aquatic Fungus Blastocladiella emersonii

Georg

Gomes

2007

Eukaryot Cell

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The global transcriptional response of the chytridiomycete Blastocladiella emersonii to environmental stress conditions was explored by sequencing a large number of expressed sequence tags (ESTs) from three distinct cDNA libraries, constructed with mRNA extracted from cells exposed to heat shock and different concentrations of cadmium chloride. A total of 6,350 high-quality EST sequences were obtained and assembled into 2,326 putative unigenes, 51% of them not previously described in B. emersonii. To approximately 59% of the unigenes it was possible to assign an orthologue in another organism, whereas 41% of them remained without a putative identification, with transcripts related to protein folding and antioxidant activity being highly enriched in the stress libraries. A microarray chip was constructed encompassing 3,773 distinct ESTs from the B. emersonii transcriptome presently available, which correspond to a wide range of biological processes. Global gene expression analysis of B. emersonii cells exposed to stress conditions revealed a large number of differentially expressed genes: 122 up-and 60 downregulated genes during heat shock and 189 up-and 110 downregulated genes during exposure to cadmium. The main functional categories represented among the upregulated genes were protein folding and proteolysis, proteins with antioxidant properties, and cellular transport. Interestingly, in response to cadmium stress, B. emersonii cells induced genes encoding six different glutathione S-transferases and six distinct metacaspases, as well as genes coding for several proteins of sulfur amino acid metabolism, indicating that cadmium causes oxidative stress and apoptosis in this fungus.Living organisms possess notable properties of adaptation to physiological changes or to adverse conditions. Unicellular organisms, in particular, must contend with fluctuations in nutrients, pH, temperature, and external osmolarity, as well as exposure to a range of potentially toxic environmental compounds. The cellular response to these environmental challenges involves drastic changes in gene expression. This reprogramming of gene transcription can be unveiled using high-throughput technologies such as expressed sequence tag (EST) sequencing and DNA microarrays, which provide valuable information about the expression patterns of the cells under determined conditions. Characterization of environmentally triggered gene expression changes provides insights into when and how each gene is expressed and offers a glimpse at the physiological response of the cells to variations in their surroundings.ESTs have historically provided data for gene discovery (18, 51), tissue-or stage-specific gene expression (3,7,14), and alternative splicing (27, 63). In fact, EST sequencing is likely to make its greatest impact on understudied genomes where little prior sequence data exist and where full genome sequencing projects may not be undertaken in the near future (36). DNA microarray technology created in the 1990s, using either whole-genome informat...

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“…S. pombe SPBC1198.08 open reading frame (named SpDUG1 hereafter) encodes a protein that has 54% identity and 71% similarity with S. cerevisiae Dug1p. We thus checked whether the SpDUG1 gene product had Cys-Gly peptidase activity, using as a tool the auxotrophy for cysteine of the cys1a⌬ strain, lacking cysteine synthase (33,34). SpDUG1 was deleted in a cys1a⌬ background strain.…”

Section: Zinc Manganesementioning

confidence: 99%

Dug1p Is a Cys-Gly Peptidase of the γ-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

Kaur

Kumar

Junot

et al. 2009

Journal of Biological Chemistry

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GSH metabolism in yeast is carried out by the ␥-glutamyl cycle as well as by the DUG complex. One of the last steps in the ␥-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the constitutent amino acids. Saccharomyces cerevisiae extracts carry Cys-Gly dipeptidase activity, but the corresponding gene has not yet been identified. We describe the isolation and characterization of a novel Cys-Gly dipeptidase, encoded by the DUG1 gene. Dug1p had previously been identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an alternate GSH degradation pathway and has also been suggested to function as a possible di-or tripeptidase based on genetic studies. We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri-or tetrapeptides in vitro. This activity requires zinc or manganese ions. Yeast cells lacking Dug1p (dug1⌬) accumulate Cys-Gly. Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We also show that the Dug1p Schizosaccharomyces pombe orthologue functions as the exclusive Cys-Gly peptidase in this organism. The human orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by complementation of the dug1⌬ mutant and by biochemical characterization, which revealed a high substrate specificity and affinity for Cys-Gly. The results indicate that the Dug1p family represents a novel class of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide (L-␥-glutamyl-L-cysteinyl-glycine) present in almost all eukaryotes (barring a few protozoa) and in a few prokaryotes (1). In the cell, glutathione exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the millimolar range), a relatively low redox potential (Ϫ240 mV), and a high stability conferred by the unusual peptidase-resistant ␥-glutamyl bond are three of the properties endowing GSH with the attribute of an important cellular redox buffer. GSH also contributes to the scavenging of free radicals and peroxides, the chelation of heavy metals, such as cadmium, the detoxification of xenobiotics, the transport of amino acids, and the regulation of enzyme activities through glutathionylation and serves as a source of sulfur and nitrogen under starvation conditions (2, 3). GSH metabolism is carried out by the ␥-glutamyl cycle, which coordinates its biosynthesis, transport, and degradation. The six-step cycle is schematically depicted in Fig. 1 (2).In Saccharomyces cerevisiae, ␥-glutamyl cyclotransferase and 5-oxoprolinase activities have not been detected, which has led to the suggestion of the presence of an incomplete, truncated form of the ␥-glutamyl cycle (4) made of ␥-glutamyl transpeptidase (␥GT) 4 and Cys-Gly dipeptidase and only serving a GSH catabolic function. Although ␥GT and Cys-Gly dipeptidase activities were detected in S. cerevisiae cell extracts, only the ␥GT gene (ECM38) has been identified so far. Cy...

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Characterization of Two Genes Encoding Putative Cysteine Synthase Required for Cysteine Biosynthesis inSchizosaccharomyces pombe

Cited by 28 publications

References 21 publications

Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

Transcriptome Analysis in Response to Heat Shock and Cadmium in the Aquatic Fungus Blastocladiella emersonii

Dug1p Is a Cys-Gly Peptidase of the γ-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

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